IntroductionLymphogranuloma venereum (LGV) is a sexually transmitted infection caused byChlamydia trachomatisgenotypes L1–L3. A combination of techniques with high discriminatory capacity such as multilocus sequence typing (MLST) and the analysis of theompAgene may be useful to determine the greater penetration of certain strains in transmission networks and their relationship with certain tropisms.AimThe aim of this study was to investigate the molecular epidemiology of LGV isolates from different regions of Spain.MethodsGenetic characterisation of LGV isolates detected in six hospitals from Spain between 2018 and 2019 was performed. MLST (five variable regions:hctB, CT058, CT144, CT172 andpbpB) andompAsequence determination were used to study the LGV strains.ResultsMost of the 161 LGV isolates (93.8%) were detected in men who have sex with men (MSM). At least 43.5% of the patients presented with HIV coinfection and 53.4% were symptomatic, with proctitis being the most prevalent symptom (73.3%). Most isolates were detected in Barcelona (n=129).The distribution ofompAgenovariants was as follows: 56.1% belonged to L2, 24.3% to L2b, 5.4% to L2bV1, 4.7% to L2bV4, 4.1% to L1, 2.7% to L2b/D-Da, 2.0% to L2bV2 and 0.7% to L2bV7. MLST was successfully performed in 81 samples and 9 different sequence types (STs) were detected. TheompAand MLST combination obtained 17 different genetic profiles, with L2-ST53 and L2-ST58 being the most prevalent (29.5% and 14.1%, respectively). L1 genotype strains belonged to ST23 (n=3) and ST2 (n=3).ConclusionLGV infections were mainly found in MSM living with HIV and with proctitis. The joint analysis ofompAand MLST genetic characterisation techniques showed a high discriminatory capacity. Our findings suggest a cocirculation of L2 and L2bompAgenotypes, and with the inclusion of MLST characterisation, the most prevalent profiles wereompAgenotype L2-MLST ST53 and L2-MLST ST58.